氧调控生丝微菌合成吡咯喹啉醌的机制研究

As the third oxidoreductase coenzyme, pyrroloquinoline quinone (PQQ) participates in electron transport of the respiratory chain and is an essential factor for the normal growth, development and reproduction of mammals. It had been considered as the fourteenth vitamin. In our previous work, a Hyphomicrobium denitrificans FJNU-R8 strain with high yield of PQQ (up to 2 g/L) was obtained, and oxygen was identified as the main control parameter for PQQ synthesis. In this study, based on our efficient PQQ producing strain, we intend to decipher the extent of oxidation state related to PQQ synthesis by introducing the oxidation-reduction potential (ORP) associated with oxygen partial pressure and pH, and establishing the coupling model between ORP and PQQ synthesis. Furthermore, we will analyze the differences in the transcriptional expression of genes involved in PQQ synthesis and methanol oxidation under various oxygen contents, to reveal the molecular mechanism of PQQ synthesis regulated by oxygen. Thirdly, important genes such as sufA and katE are designed to overexpress to enhance the synthesis of iron-sulfur cluster and the cycling of the hydrogen peroxide and oxygen, in order to alleviate the precursor condensation and the sensitivity or dependence of AHQQ oxidation on dissolved oxygen. Finally, the cytoplasmic oxidative microenvironment will be reconstituted to verify whether the catalytic activities of PqqC and PqqE are regulated by oxygen. The mechanism of oxygen-regulated PQQ synthesis will be elucidated from the perspective of cell, biochemical, and genetic level to promote the synchronization of cell growth and PQQ synthesis, shorten the fermentation cycle, and increase the production of PQQ.

吡咯喹啉醌（PQQ）作为第三种氧化还原酶辅酶，参与呼吸链的电子传递，是哺乳动物正常生长发育和繁殖的必需因子，曾被认为是第十四种维生素。我们在前期工作中选育获得一株PQQ产量高达2g/L的脱氮生丝微菌FJNU-R8，研究发现氧是其合成PQQ的主要控制参数。本项目以此为出发点，拟引入与氧分压和pH相关的氧化还原电位（ORP），建立ORP与PQQ合成的偶联模型，从ORP角度揭示PQQ合成受氧化状态的调控；分析不同氧含量下PQQ合成和甲醇氧化相关基因的转录表达差异，阐释PQQ合成受氧调控的分子基础；过表达sufA和katE等基因强化铁硫簇的合成以及过氧化氢与氧气的循环，缓解前体缩合和AHQQ氧化对溶解氧的敏感性或依赖性，重构细胞质内氧化微环境，验证氧调节PqqC和PqqE的催化活性。从细胞、生化和基因水平阐明氧调控PQQ合成的机制，促进菌体生长和PQQ合成的同步性，缩短发酵周期，提高PQQ产量。

吡咯喹啉醌（PQQ）在细胞中协同COQ10完成呼吸链电子传递，提供细胞生长所需的生物能，具有促进机体生长、线粒体增殖和清除胞内自由基等功能，在食品、医药及化妆品领域应用广泛。本项目以工业生产菌株H. denitrificans为研究对象，在前期建立的控氧培养体系基础上，分析不同pH-stat和DO-stat体系对菌体生长和PQQ合成的影响，建立DO-stat和pH-stat相结合的两阶段控氧培养工艺。5L发酵罐中，培养145 h，PQQ产量达到813.07 mg/L，单位细胞产量为15.72 mg/g DCW。进一步在培养体系中引入氧化还原电位（ORP），非线性拟合分析ORP与菌体生长和PQQ合成的相关性，培养体系ORP出现“V”字反弹，PQQ就开始大量合成，表明PQQ合成需要一个相对平衡的氧化还原微环境。因此，开发了一种三阶段ORP发酵控制工艺，50L发酵罐中培养180小时，PQQ产量达2086.07 mg/L，单位细胞产率为41.05 mg/g DCW，生产效率达11.59 mg/L/h。RT-PCR分析发现，不同溶氧条件下pqq基因簇与moxF基因簇的表达差异显著，pqqA、pqqB、pqqE和moxFα1在低溶氧条件下表达量高，pqqC、pqqD、moxFβ、moxFα2、moxFα3和moxFα4在高溶氧条件下表达量高，控氧培养能够迅速提高pqqA、pqqE、moxFα2、moxFα3和moxFβ的表达量；GO聚类及KEGG富集分析发现PQQ合成、谷氨酸和酪氨酸合成以及Fe-S簇合成等基因差异最为显著。最后，鉴定了控氧培养过程中的主要副产物（PQQ尾杂）为PQQ与甘氨酸的衍生物IPQ，设计了大孔树脂串联吸附分离的纯化工艺，实现了IPQ的高效去除。本项目证实了立项时提出的“控氧培养改变通过调节氧化还原微环境，调控pqq基因簇及其相关调控因子的表达量来影响PQQ合成”的假说。